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Bioequivalence Studies

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 1

Bioequivalence Studies
Aisha Qayyum
National University of Sciences and Technology
Pakistan

1. Introduction
During last four decades there is an increased use of generic drug products in order to lower
the healthcare cost. With increased availability and use of generic drug products, healthcare
professionals are encountered with a large number of multisource products from which they
have to select therapeutically equivalent products. Generic substitution is of concern not
only for healthcare professionals but also for pharmaceutical industries, consumers and
government officials. Many research papers have pointed out the concern regarding
standards for approval of generic products which may not always ensure therapeutic
equivalence (Boix-Montanes, 2011; Skelly, 2010; Tothfalusi et al., 2009; Midha et al., 2005;
Chen & Lesko, 2001; Chen et al., 2000; Strom, 1987; Lamy, 1986). To alleviate this fear many
guidelines/guidance and regulations covering the licensing of generic products have been
introduced to ensure that the medicinal products reaching the market have well-established
efficacy and safety profile (FDA, 1992, 1996, 2001a, 2001b, 2003, 2011; CDSCO, 2005; SFDA,
2005; Health Canada, 2004; CPMP, 2000; WHO, 1986).
Generally, demonstration of bioequivalence (BE) is the most appropriate method of
ensuring therapeutic equivalence between two medicinal products. Bioequivalence studies
should be conducted for comparison of medicinal products containing same active
substance. Such studies need to be carefully designed to take into account
biopharmaceutical, ethical, medical, pharmacokinetic, analytical and statistical
considerations. The studies should be aimed to critically assess the possibility of alternate
use of these products. In the 2003 United States Food and Drug Administration (FDA)
guidance, bioequivalence is defined as:
“the absence of a significant difference in the rate and extent to which the active
ingredient or active moiety in pharmaceutical equivalents or pharmaceutical
alternatives becomes available at the site of drug action when administered at the
same molar dose under similar conditions in an appropriately designed study” (FDA,
2003).
Bioequivalence is actually the comparison of the bioavailability of two drug products. In the
2003 United States Food and Drug Administration (FDA) guidance, bioavailability is
defined as:
“the rate and extent to which the active ingredient or active moiety is absorbed from a
drug product and becomes available at the site of action. For drug products that are not
4 Readings in Advanced Pharmacokinetics – Theory, Methods and Applications

intended to be absorbed into the blood stream, bioavailability may be assessed by

measurements intended to reflect the rate and extent to which the active ingredient or
active moiety becomes available at the site of action” (FDA, 2003).
According to World Health Organization (WHO) guidelines, bioavailability is defined as:
“the rate and extent to which the active drug ingredient or therapeutic moiety is
absorbed from a drug product and becomes available at the site of drug action” (WHO,
1986).
According to the United States Food and Drug Administration (FDA) “pharmaceutical
equivalents” are drug products that contain identical active ingredients and are identical in
strength or concentration, dosage form, and route of administration (FDA, 2011).
The CPMP (Committee for Proprietary Medicinal Products) guidance on bioavailability and
bioequivalence confers the concept of therapeutic equivalence as:
“A medicinal product is therapeutically equivalent with another product if it contains
the same active substance or therapeutic moiety and, clinically, shows the same efficacy
and safety as that product, whose efficacy and safety has been established. In practice,
demonstration of bioequivalence is generally the most appropriate method of
substantiating therapeutic equivalence between medicinal products, which are
pharmaceutically equivalent or pharmaceutical alternatives, provided they contain
excipients generally recognized as not having an influence on safety and efficacy and
comply with labeling requirements with respect to excipients. However in some cases
where similar extent of absorption but different rates of absorption are observed, the
products can still be judged therapeutically equivalent if those differences are not of
therapeutic relevance. A clinical study to prove that differences in absorption rate are
not therapeutically relevant, will probably be necessary”(CPMP, 2000).
In early 1960’s extensive work in pharmacokinetics offered substantial evidence that
composition and dosage form of a drug product can affect in vivo properties as well as
therapeutic effects. These differences have been attributed to the effect of different drug
excipients used, variations in manufacturing procedures and the properties of final dosage
form on the rate and extent of the drug absorption from its site of administration. The
importance of bioavailability came into lime-light after an incidence in Australia where a
change in an inactive excipient of phenytoin formulation by the manufacturer resulted in
low plasma levels of active drug leading to therapeutic failure and seizures in epileptic
patients who were previously well-controlled with the same dose of same drug. Similarly in
Europe marked variations in the plasma levels of digoxin were observed with different
preparations of the drug resulting in either toxicity or therapeutic failure (Crawford et al.,
2006; Welage et al., 2001; Soryal & Richens, 1992; Lindenbaum et al., 1971; Tyrer et al., 1970).
Bioequivalence and bioavailability studies are important during drug development of both
new drug products and their generic equivalents. Provision of bioavailability and/or
bioequivalence study data is an important element in support of Investigational New Drug
Applications (INDs), New Drug Applications (NDAs), Abbreviated New Drug Applications
(ANDAs) and their supplements. The term generic drug product has been defined as
“interchangeable multi-source pharmaceutical product”. Generic products are the copies of
brand-name drugs with same dosage form, strength, route of administration, intended use
Bioequivalence Studies 5

and toxicity profile as the original innovator drug. Concern about lowering healthcare costs
has resulted in an increase in the use of cheaper generic drug products instead of branded
products. The innovator drugs are protected from copying by patents that last for 20 years
from the first filing of the new chemical entity. Many people are concerned why generic
drugs are often cheaper than the brand-name versions. It is because all the development
work and clinical trials on new chemical entity are carried out by innovator to get initial
drug approval which is later on reflected in its high price whereas the generic
manufacturers only need to submit the bioequivalence data of the generic product to get a
product license. The new products need to undergo bioequivalence testing before they are
marketed. The difference may exist in absorption reflected in differing bioavailability profile
of various brands, production batches or dosage forms of a drug. This can lead to either
over- or under-medication if one entity is substituted for the other. The under-medication
can lead to therapeutic failure and on the other hand over-medication can lead to toxicity.
To avoid such risk it is best to study the bioavailability of all products but practically it is not
possible. So each drug and any change in formulation must be considered individually
while keeping in mind the real medical need for such studies in order to ensure efficacy and
safety of these drugs. Many clinicians while switching or interchanging the different
products are concerned with the safety and effectiveness of the new product. This concern is
because of the fact that small changes in bioavailability can lead to significant changes in the
efficacy or safety of the drug. Bioequivalence studies are designed with this concern in mind
and to devise the strategies that minimize the risk to the patient. So when the generic
product is pharmaceutically equivalent as well as bioequivalent to the innovator drug, then
it is expected to be therapeutically equivalent (Kowalski et al., 2006; Crawford et al., 2006;
FDA, 2003; Welage et al., 2001; Vasquez & Min, 1999; Banahan & Kolassa, 1997; Benet &
Goyan, 1995; Marzo and Balant, 1995; WHO, 1986).

2. Design and conduct of bioequivalence studies

The basis of a bioequivalence study is the comparison of the drug product to be tested with
an appropriate reference product (branded innovator drug). In bioequivalence studies an
applicant compares the systemic exposure profile of a test drug to that of a reference drug
product. Bioequivalence of two products can be assessed using in vitro standards,
pharmacokinetic profile, clinical or pharmacodynamic end points. Different approaches for
determination of bioequivalence of a drug product are:
 An in vivo test in humans in which the concentration of the active ingredient and when
appropriate, its active metabolites, in blood, plasma, serum or other suitable biological
fluid is measured as a function of time.
 An in vivo test in humans in which the urinary excretion of the active ingredient and
when appropriate, its active metabolites are measured as a function of time.
 An in vitro test that has been correlated with and is predictive of human bioavailability
profile or the one acceptable to FDA (e.g. dissolution rate test) that ensures human in
vivo bioavailability.
 An in vivo test in humans in which an appropriate pharmacological effect of the active
ingredient and when appropriate, its active metabolites are measured as a function of
time if this effect can be measured with adequate accuracy, sensitivity and
reproducibility.
6 Readings in Advanced Pharmacokinetics – Theory, Methods and Applications

 Well-controlled clinical trials that establish the efficacy and safety of the drug product,
for purpose of determining bioavailability, or comparative clinical trials, for purpose of
demonstrating bioequivalence.
 Any other approach considered adequate by the FDA to measure bioavailability or
ascertain bioequivalence.
Bioequivalence for most of oral tablets or capsules is demonstrated in vivo by comparing the
rate and extent of absorption that is bioavailability of the generic product with that of the
innovator product. This is done by measuring the active ingredient concentration in blood,
plasma, serum or other biological fluids over a certain period of time for both the generic
and innovator products, also called test and reference drugs respectively. By doing so the
bioequivalence studies frequently rely on pharmacokinetic measures such as area under the
concentration-time curve (AUC) and peak drug concentration (Cmax) (Niazi, 2007; FDA,
2001a, 2003; Pidgen, 1996; Nation & Sanson, 1994).

2.1 Study design

Many authors have debated whether multi-dose or single-dose studies should be used to
assess bioequivalence. Generally single-dose pharmacokinetic studies are recommended
for both immediate- and modified-release drug products as they are more sensitive in
assessing the active ingredient released from drug into circulation. For assessing
bioequivalence of two formulations of a drug, two-sequence, two-period, crossover study
is conducted after administration of single dose under fasted conditions. In crossover
design the subjects serve as their own controls and they crossover from one treatment to
the other. A large variability in drug clearance often exists among the individuals.
However the intrasubject variation is usually smaller relative to inter-subjects variability.
Parallel studies are appropriate if the drug has extremely long half life, repeated
pharmacokinetic profile is difficult to obtain, or residual pharmacodynamic effects are
relevant. Furthermore, if carry over effects from one treatment period to another are of
concern or if intrasubject variability is high, then replicated design is used. Nonreplicate
study designs are usually recommended for bioequivalence studies of most of the orally
administered, modified-release and immediate-release dosage forms. Replicate study
designs are often recommended for bioequivalence studies of highly variable drug
products (intra-subject coefficient of variation ≥ 30%), including those that are modified-
release, immediate release, and other orally administered drug products. Replicate study
designs have several scientific advantages compared to nonreplicate designs. (SFDA,
2005; FDA, 2001a, 2003; Welage et al., 2001; Nation & Sanson, 1994; Steinijans et al., 1992;
Metzler, 1989).

2.2 Study subjects

The subjects should be selected with the objective of minimizing variability and
permitting detection of difference between the drug products. Therefore, the study is
normally carried out with healthy subjects. The study is performed in accordance with the
Declaration of Helsinki for biomedical research involving human subjects (WMA
Declaration of Helsinki, 2008) and the Guideline for Good Clinical Practice (FDA, 1996).
The subjects recruited for bioequivalence studies should be 18 years of age or older and
Bioequivalence Studies 7

capable of giving informed consent. Generally adults between 20-40 years should be
selected. According to FDA guidance and Canadian and European guidelines a minimum
of 12 subjects are recruited for bioequivalence studies. For logistic reasons the total
number normally does not exceed 24 subjects. The subjects should be in good health. The
subject’s health is assessed by medical examination including medical history and
laboratory tests. They should be screened for the history of use of medications or drugs of
abuse, alcohol intake and smoking. The subjects should not take any medication one week
before start of study (CDSCO, 2005; FDA, 2001a, 2003; Marzo & Balant, 1995; Nation &
Sanson, 1994; WHO, 1986).

2.3 Drug administration and sampling

A bioequivalence study should be a single dose comparison of test drug with appropriate
reference drug product carried out in healthy adults. The drug is administered to the
subjects in fasting state, unless some other approach is more suitable for valid scientific
reasons. Co-administration of food with oral drugs may either enhance or interfere with
drug absorption. Thus, feeding increases the inter- and intra-subject variations in rate and
extent of absorption. The sponsor should provide the rationale for conducting
bioequivalence study under fed or fasting conditions. The subjects are randomly selected
for each group in the study and the sequence of drug administration is randomly assigned
to the individuals. In a typical situation of comparing a test formulation (T) with a
reference formulation (R), the two-period, two-sequence crossover design is the RT/TR
design as shown in table 1. Subjects are randomly allocated to two treatment sequences; in
sequence 1, subjects receive the reference drug and test drug in periods 1 and 2
respectively, on the other hand in sequence 2, subjects receive the drug products in
reverse order. The administration of each product is followed by a sufficiently long wash
out period of time to ensure complete elimination of drug before next administration. A
time period of more than 5 half-lives of the drug is considered adequate washout period.
In selected cases, it may be necessary for the test and reference products to be compared
after multiple-dose administration to determine steady-state levels of the active drug
moiety. A multiple-dose study should be crossover in design, unless a parallel or other
design is more suitable for valid scientific reasons (Hauschke et al., 2007; Niazi, 2007;
FDA, 2003; Makoid et al., 1999).
In fasted state studies an overnight fast of at least 10 hours is recommended. Generally in
single dose studies the highest marketed strength is administered. The doses of the test
and reference products should be same. The test or reference products are administered
with 240 ml of water. Liquids are allowed after one hour and standard meal after 4 hours
of drug administration. In all the studies the standardization of study environment, diet,
fluid intake and exercise is important (CDSCO, 2005; FDA, 2003; WHO, 1986).

Sequence Period 1 Washout Period 2

1 R T
2 T R
Table 1. RT/TR Design
8 Readings in Advanced Pharmacokinetics – Theory, Methods and Applications

Under most of the conditions blood or plasma is collected rather than urine or tissue. Blood
samples are drawn at appropriate times to assess the absorption, distribution and
elimination phases of the drug. For most of the drugs 12-18 samples are recommended
including pre-dose sample from each subject. Generally sampling for a period equal to at
least 3 times the terminal half life of the drug is recommended. Other approach is that the
duration of sampling should be sufficient to define at least 80% of the total area under the
concentration–time curve (AUC). The exact timings for sampling depend on nature and
pharmacokinetic profile of individual drug and its dosage form (FDA, 2001a, 2003; Nation &
Sanson, 1994; WHO, 1986).

2.4 Bioanalytical methodology

The measurement of drug concentration in collected samples is done through bioanalytical
methods. Prior to sample analysis, the selected analytical method is validated in accordance
with the recommended guidelines (Niazi, 2007; ICH, 2005; FDA, 2001b). Assay validation
involves different steps:
 Quality control samples
 Identification and specificity
 Sensitivity and limit of detection
 Range, linearity and limit of quantitation
 Precision and accuracy
 Analyte and system stability
 Reproducibility
A properly validated assay method is crucial for the acceptance of any pharmacokinetic
study. During validation, quality control samples are run in replicates to assess the intra-
and inter-day variability during sample analysis.

2.5 Data analysis

Data analysis is carried out:

 By direct observation and measurement

 By simple mathematical calculations
 By use of different softwares

2.5.1 Pharmacokinetic analysis

Pharmacokinetic analysis is done using the blood or plasma concentration-time profile. The
pharmacokinetic parameters to be measured depend on the type of study whether single-
dose or multiple-dose study (FDA, 1992).
For single dose bioequivalence study the parameters are:

 Area under the plasma / blood concentration-time curve from time zero to
time t (AUC0-t), calculated by trapezoidal rule, where t is the last measurable time
point.
Bioequivalence Studies 9

 Area under the plasma / blood concentration-time curve from time zero to time infinity
(AUC0–∞) where

AUC0–∞ = AUCt + Ct / λz
Ct is the last measurable drug concentration and λz is the terminal elimination rate
constant calculated according to an appropriate method. The terminal or elimination
half life of the drug should also be documented.
 Peak drug concentration (Cmax) and the time to peak drug concentration (Tmax),
obtained directly from the data without interpolation.
For multiple-dose studies, the parameters measured are:

 Area under the plasma / blood concentration-time curve from time zero to time  over
a dosing interval at steady state (AUC0-), where  is the dosing interval.
 Peak drug concentration (Cmax) and the time to peak drug concentration (Tmax),
obtained directly from the data without interpolation, after the last dose is
administered.
 Drug concentrations at the end of each dosing interval during steady state (Cmin).
 Average drug concentration at steady state (Cav), where Cav = AUC0- /.
 Degree of fluctuation (DF) at steady state, where DF = 100% × (Cmax — Cmin) / Cav.

2.5.2 Statistical analysis

The pharmacokinetic parameters AUC and Cmax are analyzed statistically to determine if
the test and reference products produce comparable values. The FDA’s statistical criteria
for approval of test or generic drugs requires calculation of a confidence interval (CI) for
the ratio between the means of test and reference product’s pharmacokinetic variables.
The two products are said to be bioequivalent if the 90% CI for the ratio of test to
reference formulation falls within the bioequivalence acceptance range of 80-120% for
data in original scale and 80-125% for log-transformed data of AUC and Cmax. This
method is equivalent to a testing procedure called two one-sided tests (TOST) procedure,
where one test verifies that the bioavailability of the test product is not too low and the
other to show that it is not too high as compared to standard reference product. The
current practice is to carry out two one-sided tests (TOST) procedure with the null
hypothesis (H0) of non-bioequivalence at 5% level of significance (α=0.05). Traditional
statistical approach is often designed to test the null hypothesis of equality. If data is
sufficiently strong, null hypothesis is rejected and alternate hypothesis (H1) is accepted.
Before 1980s, most of the bioequivalence studies were conducted in this way; researchers
tested for differences between drug formulations and if they found none, they concluded
them to be bioequivalent (i.e. H0 = bioequivalence, H1 = non-bioequivalence). During
further studies, many flaws were recognized in this approach. If sample size was large
enough, minor differences even not important clinically, were found to be significant,
whereas if sample size was small, the potential important differences were neglected.
The purpose of bioequivalence (BE) study is generally not to demonstrate a difference but
to assess the equivalence of test product to that of reference standard. So the method of
difference statistics with null hypothesis of no difference is not applicable to BE studies.
10 Readings in Advanced Pharmacokinetics – Theory, Methods and Applications

Instead, the equivalence testing with the null hypothesis of a difference or non-
bioequivalence is used. According to the FDA this difference is set at –20 / +25 percent.
In order to verify that –20 / +25 percent rule is satisfied, the two one-sided tests are
carried out. The rejection of the two one-sided tests null hypotheses at 5% level of
significance (α=0.05) is equivalent to the inclusion of the 90 percent CI in the acceptance
range (Hauschke et al., 2007; Riffenburgh, 2006; Welage et al., 2001; FDA, 1992, 2001a;
Pidgen, 1996; Hauck & Anderson, 1992; Schuirmann, 1987).
The statistical analysis ANOVA (analysis of variance) is used to calculate estimates of the
error variance. ANOVA should be performed on AUC and Cmax accounting for the sources
of variation which are:
 Sequence (group)
 Subjects in a sequence
 Period (phase)
 Treatment (drug formulation).
The results of ANOVA are calculated at 5% level of significance (α=0.05). The sponsor may
use untransformed or log-transformed data. The choice should be made with concurrence
by the FDA prior to conducting the study. The validity of statistical analysis is improved by
log-transforming the raw data prior to analysis (FDA, 1992).

2.6 Presentation and documentation of data

The drug concentration in the biological fluid at each sampling time point for all the subjects
should be presented in original form. Pharmacokinetic parameters like Cmax, Cmin, Tmax, are
directly observed from original data. Pharmacokinetic parameters like AUC0-t, AUC0–∞,
AUC0-, λz, t1/2 are derived from original data by mathematical calculations or by using
different softwares like APO MWPHARM, PK Solutions, PK-fit and WinNonlin PK
software. The pharmacokinetic data recommended for submission is:
 Plasma concentrations and time points
 Subject, period, sequence, treatment
 AUC0-t, AUC0–∞, λz, t1/2, Cmax and Tmax
 AUC0-, Cmin, Cav and degree of fluctuation are also submitted for multiple-dose studies
 Intersubject, intrasubject, and/or total variability
The mean values and standard deviation (SD) can be calculated by computer programs like
Microsoft Excel, SAS, SPSS. The statistical analysis for bioequivalence testing is carried out
by using different computer softwares like EquivTest 2.0, Minitab Release 13.1, BioEquiv
and DAS 2.0 software. The statistical information recommended to be provided for
pharmacokinetic parameters are:
 Geometric mean
 Arithmetic mean
 Ratio of means
 Confidence interval
Bioequivalence parameters can be presented in tabulated form as shown in table 2.
Bioequivalence Studies 11

90 percent
Test Reference Ratio of
Parameters confidence
mean ± SD mean ± SD Geometric means
interval
AUC(0-t)
(hr.mg/l)
AUC(0-∞)
(hr.mg/l)
Cmax (mg/l)
Tmax (hrs)
Table 2. Bioequivalence Parameters

3. Waivers of in vivo bioequivalence studies

Under certain circumstances, FDA may waive the requirement for in vivo bioequivalence
studies if drug product meets one of the following criteria:
 When the drug product is a parentral solution intended solely for administration by
injection, and contains the active drug ingredient in the same solvent and concentration
as a solution that is subject of an approved full New Drug Application (NDA).
 The drug product is a topically applied preparation intended for local therapeutic effect
e.g. ophthalmic/otic solutions or it is administered by inhalation and contains the active
drug ingredient in the same dosage form as a drug product that is the subject of an
approved full NDA and ANDA.
 The drug product is a solution for application to the skin, an oral solution, elixir, syrup,
tincture, a solution for nebulization, a nasal solution, or similar other solubilized form,
and contains an active drug ingredient in the same concentration and dosage form as a
drug product that is the subject of an approved full NDA or ANDA, and contains no
inactive ingredient or other change in formulation from the drug product that is the
subject of an approved full NDA and ANDA that may significantly affect absorption of
the active drug ingredient or moiety for products that are systemically absorbed, or that
may significantly affect systemic or local availability for products intended to act
locally.
 The drug product is a solid oral dosage form (other than controlled release or enteric-
coated) that has been determined to be effective for at least one indication in a Drug
Efficacy Study Implementation (DESI) notice and is not included in the FDA list of
drugs for which in vivo bioequivalence testing is required.
 The in vivo bioavailability or bioequivalence may be self-evident for certain drug
products. The FDA may waive the requirement for the submission of evidence obtained
by in vivo measuring the bioavailability or demonstrating the bioequivalence of these
drug products. A drug product’s in vivo bioavailability or bioequivalence may be
considered self-evident based on other data in the application.
 For certain drug products, bioavailability or bioequivalence may be demonstrated by
evidence obtained in vitro in lieu of in vivo data. The FDA may waive the requirement of
the submission of in vivo data if a drug product meets the following criteria:
12 Readings in Advanced Pharmacokinetics – Theory, Methods and Applications

 The drug product is in the same dosage form, but in the different strength, and is
proportionally similar in its active and inactive ingredients to another drug product
for which the same manufacturer has got the approval and certain conditions are
met, that the bioavailability of this other drug product has been measured and both
meet an appropriate in vitro test approved by the FDA; and the applicant submits
evidence showing that both products are proportionally similar in their active and
inactive ingredients.
 The drug product is shown to meet an in vitro test that assures bioavailability, that
in vitro test has been correlated with in vivo data.
 The drug product, for which only an in vitro bioequivalence data has been required
by FDA for approval.
 The drug product is a reformulated product that is identical, except for a different
color, flavor, or preservative, to another drug product for which the same
manufacturer has obtained approval and the following conditions are met: the
bioavailability of the other product has been measured; and both the drug products
meet an appropriate in vitro test approved by the FDA.
In above circumstances bioequivalence studies may be waived by the drug regulatory
authorities (FDA, 2011, 2000, 2003; Niazi, 2007; CDSCO, 2005; Makoid et al., 1999).

4. Conclusion
Keeping in view the health-care cost, the pharmaceutical companies are manufacturing and
marketing cheaper generic drug products. It is vital for the regulatory authorities of every
country to ensure the efficacy and safety of these generic formulations. Carefully planned
and designed bioequivalence studies are the only way to ensure uniformity in standards of
quality, efficacy and safety of pharmaceutical products.

5. Acknowledgement
The author thanks Prof M. H. Najmi (Professor of Pharmacology and Therapeutics
Foundation Medical University) and Prof Muhammad Nawaz (Vice Chancellor of
University of Veterinary and Animal Sciences) for their guidance and inspiration. The
author appreciates the financial assistance provided by National University of Sciences and
Technology (NUST) for producing this piece of work.

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14 Readings in Advanced Pharmacokinetics – Theory, Methods and Applications

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